The present invention relates to a novel human gene encoding a polypeptide which is a member of the chemokine family. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named Chemokine Alpha-6, hereinafter referred to as xe2x80x9cCKxcex1-6xe2x80x9d. CKxcex1-6 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the immune system, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying agonists and antagonists of CKxcex1-6 activity.
The ability to control the migration and xe2x80x9ctraffickingxe2x80x9d of various cell types is controlled by a subset of factors, or proteins, among which chemokines are an example.
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related chemotactic cytokines. These molecules are usually 8-10 kd in size. In general, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the xe2x80x9cC-X-Cxe2x80x9d subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the xe2x80x9cCxe2x80x94Cxe2x80x9d subfamily. Thus far, at least sixteen different members of this family have been identified in humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
Members of the xe2x80x9cCxe2x80x94Cxe2x80x9d branch exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophages which suppress tumor formation in vertebrates; and basophils which release histamine which plays a role in allergic inflammation. However, members of one branch may exert an effect on cells which are normally responsive to the other branch of chemokines and, therefore, no precise role can be attached to the members of the branches.
While members of the Cxe2x80x94C branch act predominantly on mononuclear cells and members of the C-X-C branch act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline. Some chemokines from one family show characteristics of the other.
The polypeptide of the present invention has the conserved cysteine residues of the xe2x80x9cC-X-Cxe2x80x9d region, and has amino acid sequence homology to known chemokines.
Thus, there is a need for polypeptides that function as regulators of the migration of distinct cell types and of their roles in dysfunction and disease, since disturbances of such regulation may be involved in disorders relating to hemostasis, angiogenesis, tumor metastisis, cellular migration and ovulation, as well as neurogenesis. Therefore, there is a need for identification and characterization of such human polypeptides which can play a role in detecting, preventing, ameliorating or correcting such disorders.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the CKxcex1-6 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the human cDNA in clone xe2x80x9cHFCET92xe2x80x9d deposited as plasmid DNA as ATCC(copyright) Deposit Number 209300 on Sep. 25, 1997 and as ATCC(copyright) Deposit Number 209643 on Feb. 25, 1998. The nucleotide sequence determined by sequencing the deposited CKxcex1-6 clone (HFCET92), which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 84 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 46-48. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in SEQ ID NO:2, or the complete amino acid sequence excepting the N-terminal methionine encoded by a cDNA clone HFCET92, which molecules also can encode additional amino acids fused to the N-terminus of the CKxcex1-6 amino acid sequence.
The polypeptide of the present invention has amino acid sequence homology to known chemokines, including the conserved C-X-C cysteine pattern characteristic of the alpha subfamily of chemokines beginning with the cysteine at position 22 in SEQ ID NO:2.
CKxcex1-6 also lacks the ELR motif found in some alpha chemokines immediately preceding the first cysteine residue, which is known to be required for the neutrophil and endothelial cell chemotactic activity as well as the angiogenic activity of IL-8.
The encoded polypeptide has a predicted leader sequence of about 16, 17, 18, 19, 20 or 21 amino acids (the first 16 of which are underlined in FIG. 1); and the amino acid sequence of the predicted mature CKxcex1-6 proteins are also shown in FIG. 1 as amino acid residues 17-84, 18-84, 19-84, 20-84, 21-84, and 22-84 (SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the CKxcex1-6 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding the CKxcex1-6 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2) excepting the N-terminal methionine (i.e., positions 2 to 84 of SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted mature CKxcex1-6 polypeptide having the amino acid sequence at positions 16-84, 17-84, 18-84, 19-84, 20-84, 21-84 or 22-84 in FIG. 1 (SEQ ID NO:2); (d) a nucleotide sequence encoding a biologically active fragment of the polypeptide of (a); (e) a nucleotide sequence encoding the CKxcex1-6 polypeptide having the complete amino acid sequence encoded by the human cDNA in clone HFCET92; (f) a nucleotide sequence encoding the CKxcex1-6 polypeptide having the complete amino acid sequence excepting the N-terminal methionine encoded by the human cDNA in clone HFCET92; (g) a nucleotide sequence encoding a mature CKxcex1-6 polypeptide encoded by the human cDNA in clone HFCET92; (h) a nucleotide sequence encoding a biologically active fragment of the polypeptide of (e); and (i) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g) or (h), above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h) or (i), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a CKxcex1-6 polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g) or (h), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of CKxcex1-6 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated CKxcex1-6 polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the full-length CKxcex1-6 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the human cDNA in clone HFCET92; (b) the amino acid sequence of the full-length CKxcex1-6 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions 2 to 84 of SEQ ID NO:2) or the complete amino acid sequence excepting the N-terminal methionine encoded by the human cDNA in clone HFCET92; (c) the amino acid sequence of the mature CKxcex1-6 polypeptide having the amino acid sequence shown in SEQ ID NO:2 as residues 17-84, 18-84, 19-84, 20-84, 21-84 or 22-84 or the amino acid sequence of a mature CKxcex1-6 polypeptide encoded by the human cDNA in clone EFCET92; and (d) the amino acid sequence of a biologically active fragment of the polypeptide of (a). The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those described in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity, to those above. Polynucleotides encoding such polypeptides are also provided.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-bearing portion of a CKxcex1-6 polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a CKxcex1-6 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
In another embodiment, the invention provides an isolated antibody that binds specifically to a CKxcex1-6 polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above. The invention further provides methods for isolating antibodies that bind specifically to a CKxcex1-6 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The invention also provides for pharmaceutical compositions comprising CKxcex1-6 polypeptides, particularly human CKxcex1-6 polypeptides, which may be employed, for instance, to stimulate wound healing, to treat solid tumors, microbial infections, autoimmune diseases, liver cirrhosis, osteoarthritis, to stimulate neural growth and to treat pulmonary fibrosis. Methods of treating individuals in need of CKxcex1-6 polypeptides are also provided.
The invention further provides compositions comprising a CKxcex1-6 polynucleotide or a CKxcex1-6 polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a CKxcex1-6 polynucleotide for expression of a CKxcex1-6 polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of a CKxcex1-6.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of the CKxcex1-6 polypeptide, which involves contacting a receptor polypeptide which is enhanced by the CKxcex1-6 polypeptide with the candidate compound in the presence of a CKxcex1-6 polypeptide, assaying calcium mobilization or chemotactic activity of the cell expressing the receptor in the presence of the candidate compound and of CKxcex1-6 polypeptide, and comparing the receptor activity to a standard level of activity, the standard being assayed when contact is made between the receptor and the CKxcex1-6 polypeptide in the absence of the candidate compound. In this assay, an increase in calcium mobilization or chemotaxis over the standard indicates that the candidate compound is an agonist of CKxcex1-6 activity and a decrease in calcium mobilization or chemotaxis compared to the standard indicates that the compound is an antagonist of CKxcex1-6 activity.
It has been discovered that CKxcex1-6 is expressed in cDNA libraries derived from adult cerebellum, fetal brain, rejected kidney and bone marrow tissues. Therefore, nucleic acids of the invention are useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to those polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). In addition, for a number of disorders of the above tissues or cells, particularly of the immune system, significantly higher or lower levels of CKxcex1-6 gene expression may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d CKxcex1-6 gene expression level, i.e., the CKxcex1-6 expression level in healthy tissue from an individual not having the immune system disorder. Thus, the invention provides a diagnostic method useful during diagnosis of such a disorder, which involves: (a) assaying CKxcex1-6 gene expression level in cells or body fluid of an individual; (b) comparing the CKxcex1-6 gene expression level with a standard CKxcex1-6 gene expression level, whereby an increase or decrease in the assayed CKxcex1-6 gene expression level compared to the standard expression level is indicative of disorder in the immune system.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of CKxcex1-6 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated CKxcex1-6 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of CKxcex1-6 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a CKxcex1-6 antagonist. Preferred antagonists for use in the present invention are CKxcex1-6-specific antibodies.